Frequency Of DNA Strand Breaks Research Articles

Association of DNA damage with vitamin D and hair heavy metals of obese women

BackgroundObesity has been linked to DNA damage. The modifiable risk factors may modulate the impact of obesity on DNA damage.ObjectiveThis study aimed to assess DNA damage and its association with dietary nutrient, serum 25-hydroxyvitamin D (25(OH)D) and concentration of hair heavy metals of obese and non-obese women.MethodA case–control study was conducted involving 134 women aged between 20 and 50 years. Serum 25(OH)D, fasting glucose, and lipid profile were assessed. Indicators of DNA damage such as percentage of tail DNA, tail moment, tail olive moment, tail intensity and tail length were measured using an alkaline-comet assay. Concentrations of hair heavy metals were quantified using inductively coupled plasma–mass spectrometry (ICP–MS). Participants' daily energy, macro, and micronutrient intake were collected using the Food Frequency Questionnaire.ResultsMean values of serum 25(OH)D was 31.8 ± 0.9 nmol/L. 96.3% of participants were vitamin D deficiency (< 50 nmol/L). The mean BMI was 26.3 ± 0.5 kg/m2. Half of the participants (50.7%) have a high frequency of DNA strand breaks. Mean concentration of hair heavy metals (mg/kg) were 0.1 ± 0.03 (arsenic), 0.2 ± 0.1 (cadmium), 1.0 ± 0.4 (mercury), 2.8 ± 0.8 (lead),and 6.2 ± 0.4 (chromium). There was no significant difference for the mean of serum 25(OH)D, indicators of DNA damage, concentrations of hair heavy metals and dietary nutrients between obese and non-obese groups (p > 0.05). Obese women with serum 25(OH)D level of ≥ 31 nmol/L had a significantly lower tail moment (p = 0.029) and tail olive moment (p = 0.031); thus, indicating less DNA damage. Additionally, obese women with hair chromium concentration of ≥ 5.88 mg/kg had a significantly higher tail moment (p = 0.047), indicating more DNA damage.ConclusionDNA damage among obese women correlated with serum 25(OH)D and hair chromium.

Exploring the sublethal genotoxic effects of class II organophosphorus insecticide quinalphos on freshwater fish Cyprinus carpio

The genotoxic effects of quinalphos (QP) were evaluated in the erythrocytes of Cyprinus carpio using the comet, micronucleus, and nuclear abnormality assays. The 96 h LC50 value of quinalphos, estimated by static renewal system, was 10.9 µL/L. On the basis of the LC50 value, two sublethal concentrations (1.09 and 2.18 µL/L) of the compound were used together with cyclophosphamide (5 mg/L) as the positive control. The fish were exposed for 35 d, and sampling was done at the end of 7, 14, 21, 28, and 35 d. Our results reveal a significant dose-dependent increase in the frequencies of DNA strand breaks, micronucleus, and nuclear abnormalities. Similar responses were also observed in fish exposed to the positive genotoxin cyclophosphamide. The collective findings demonstrated that quinalphos exerts a genotoxic effect on C. carpio. The results further confirmed that the comet, micronucleus, and nuclear abnormality assays are useful tools in determining the potential genotoxicity of pesticides towards aquatic organisms.

Polymethoxy-1-Alkenes Screening of Chlorella and Spirulina Food Supplements Coupled with In Vivo Toxicity Studies.

Selected species of cyanobacteria and green algae have been reported to produce lipophilic polymethoxy-1-alkenes (PMAs) which were shown to exhibit in vivo teratogenicity. Considering that information on PMAs in Arthospira sp. (known commercially as Spirulina) and Chlorella sp. cultivated for food supplement production was essentially lacking, the present study screened Chlorella (n = 10) and Spirulina (n = 13) food supplements registered in the European Union. Mass spectrometry analysis of column fractionated extracts was performed. None of the four variants previously reported in some cyanobacteria and green algae, nor any potentially related structures were detected in the studied samples. Since the isolated lipophilic fractions contained various compounds, they were further screened for in vivo teratogenicity in Danio rerio embryo, and for the potential to induce oxidative stress and genotoxicity in the liver and neurotoxicity in the brain of adult zebrafish. None of the tested food supplements had detectable levels of PMAs or any potentially related structures. No teratogenicity was revealed except for spinal curvature induced by fractions obtained from two Chlorella products. Selected fractions revealed cytotoxicity as indicated by an increased level of reactive oxygen species, catalase activity, lipid peroxidation and increased frequency of DNA strand breaks in hepatic tissue. The majority (60%) of Chlorella fractions induced an increase in cholinesterase activity in zebrafish brain homogenate while exposure to 61.5% of Spirulina fractions was associated with its decrease. The present study confirms that Chlorella and Spirulina food supplements are free of teratogenic PMAs, although the observed in vivo toxicities raise questions regarding the quality of selected products.

Increased reactive oxygen species and exhaustion of quiescent CD34-positive bone marrow cells may contribute to poor graft function after allotransplants.

Poor graft function (PGF) is a fatal complication following allogeneic haematopoietic stem cell transplantation. However, the underlying mechanism is unclear. Effective cross-talk between haematopoietic stem cells (HSCs) and bone marrow microenvironment is important for normal haematopoiesis. Normal HSCs reside in a hypoxic bone marrow microenvironment that protects them from oxidative stress that would otherwise inhibit their self-renewal and results in bone marrow failure. Whether an increased level of reactive oxygen species (ROS) causes PGF following allotransplant is unclear. Using a prospective case-pair study, we identified increased levels of ROS in CD34+ bone marrow cells in subjects with PGF. Elevated ROS levels was associated with an increased frequency of DNA strand breaks, apoptosis, exhaustion of quiescent CD34+ cells and defective colony-forming unit plating efficiency, particularly in the CD34+CD38− fraction. Up-regulated intracellular p53, p21, caspase-3 and caspase-9 levels (but not p38) were detected in CD34+ cells, particularly in the CD34+CD38− fraction. To further study the potential role of ROS levels in post-transplant haematopoiesis, CD34+ bone marrow cells from subjects with good graft function were treated with H2O2. This increased ROS levels resulting in defective CD34+ cells, an effect partially reversed by N-acetyl-L-cysteine. Moreover, CD34+ bone marrow cells from the donors to subjects with poor or good graft function exhibited comparable haematopoietic reconstitution capacities in the xeno-transplanted NOD-PrkdcscidIL2rgnull mice. Thus, even if the transplanted donors' bone marrow CD34+ cells are functionally normal pre-transplant, ROS-induced apoptosis may contribute to the exhaustion of CD34+ bone marrow cells in subjects with PGF following allotransplant.

Genotoxic and teratogenic effect of freshwater sediment samples from the Rhine and Elbe River (Germany) in zebrafish embryo using a multi-endpoint testing strategy.

The embryotoxic potential of three model sediment samples with a distinct and well-characterized pollutant burden from the main German river basins Rhine and Elbe was investigated. The Fish Embryo Contact Test (FECT) in zebrafish (Danio rerio) was applied and submitted to further development to allow for a comprehensive risk assessment of such complex environmental samples. As particulate pollutants are constructive constituents of sediments, they underlay episodic source-sink dynamics, becoming available to benthic organisms. As bioavailability of xenobiotics is a crucial factor for ecotoxicological hazard, we focused on the direct particle-exposure pathway, evaluating throughput-capable endpoints and considering toxicokinetics. Fish embryo and larvae were exposed toward reconstituted (freeze-dried) sediment samples on a microcosm-scale experimental approach. A range of different developmental embryonic stages were considered to gain knowledge of potential correlations with metabolic competence during the early embryogenesis. Morphological, physiological, and molecular endpoints were investigated to elucidate induced adverse effects, placing particular emphasis on genomic instability, assessed by the in vivo comet assay. Flow cytometry was used to investigate the extent of induced cell death, since cytotoxicity can lead to confounding effects. The implementation of relative toxicity indices further provides inter-comparability between samples and related studies. All of the investigated sediments represent a significant ecotoxicological hazard by disrupting embryogenesis in zebrafish. Beside the induction of acute toxicity, morphological and physiological embryotoxic effects could be identified in a concentration-response manner. Increased DNA strand break frequency was detected after sediment contact in characteristic non-monotonic dose-response behavior due to overlapping cytotoxic effects. The embryonic zebrafish toxicity model along with the in vivo comet assay and molecular biomarker analysis should prospectively be considered to assess the ecotoxicological potential of sediments allowing for a comprehensive hazard ranking. In order to elucidate mode of action, novel techniques such as flow cytometry have been adopted and proved to be valuable tools for advanced risk assessment and management.

Dicarbonyl proteome and genome damage in metabolic and vascular disease

Methylglyoxal is a potent protein-glycating agent. It is an arginine-directed glycating agent and often modifies functionally important sites in proteins. Glycation forms mainly MG-H1 [Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine] residues. MG-H1 content of proteins is quantified by stable isotopic dilution analysis-MS/MS and also by immunoblotting with specific monoclonal antibodies. Methylglyoxal-modified proteins undergo cellular proteolysis and release MG-H1 free adduct for excretion. MG-H1 residues have been found in proteins of animals, plants, bacteria, fungi and protoctista. MG-H1 is often the major advanced glycation end-product in proteins of tissues and body fluids, increasing in diabetes and associated vascular complications, renal failure, cirrhosis, Alzheimer's disease, arthritis, Parkinson's disease and aging. Proteins susceptible to methylglyoxal modification with related functional impairment are called the DCP (dicarbonyl proteome). The DCP includes albumin, haemoglobin, transcription factors, mitochondrial proteins, extracellular matrix proteins, lens crystallins and others. DCP component proteins are linked to mitochondrial dysfunction in diabetes and aging, oxidative stress, dyslipidaemia, cell detachment and anoikis and apoptosis. Methylglyoxal also modifies DNA where deoxyguanosine residues are modified to imidazopurinone MGdG {3-(2'-deoxyribosyl)-6,7-dihydro-6,7-dihydroxy-6/7-methylimidazo-[2,3-b]purine-9(8)one} isomers. MGdG was the major quantitative adduct detected in vivo. It was linked to frequency of DNA strand breaks and increased markedly during apoptosis induced by a cell-permeant glyoxalase I inhibitor. Glyoxalase I metabolizes >99% methylglyoxal and thereby protects the proteome and genome. Gene deletion of GLO1 is embryonically lethal and GLO1 silencing increases methylglyoxal concentration, MG-H1 and MGdG, premature aging and disease. Studies of methylglyoxal glycation have importance for human health, longevity and treatment of disease.

Changes of Reactive Oxygen and Nitrogen Species and Mitochondrial Functioning in Human K562 and HL60 Cells Exposed to Ionizing Radiation

Free radicals generated by mitochondria are candidates for mediating long-lasting effects of radiation on cells, including genetic instability. To better understand the significance of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in these long-term effects we assayed ROS and RNS levels, the mitochondrial membrane potential and mass, and the frequency of DNA strand breaks, apoptosis and necrosis in human leukemic cells (K562 and HL60) after 12 Gy of X irradiation. An increase in intracellular ROS level was observed immediately post-irradiation, and about 24 h later a second increase of ROS was accompanied by increase in nitrogen oxide, mitochondrial potential and mitochondrial mass in both cell types. The second peak of ROS level was partially inhibited by rotenone, an inhibitor of mitochondrial complex I, in K562 but not in HL60 cells suggesting that the sources of ROS differed in the two cell types. The frequency of DNA breaks showed kinetics similar to ROS levels, with a sharp peak immediately after irradiation and a second increase 24 and 48 h later, which was significantly higher in K562 cells. Forty-eight hours after irradiation an increase in the frequency of apoptotic cells was observed in both cell lines, which became larger and statistically significant in K562 cells after inhibition of mitochondrial complex I. Our results show that ionizing radiation activates cellular processes which produce long-lasting ROS and RNS radicals, which may have different sources in different cell types and could participate in cellular signaling networks important for radiosensitivity and mode of cell death.

Evaluation of oxidative stress and genetic damage caused by detergents in the zebrafish Danio rerio (Cyprinidae)

Detergents are used in large quantities and some of their ingredients are highly toxic to aquatic organisms. In the present study the toxicity (lipid peroxidation) and genotoxic (frequency of DNA strand breaks) effects were evaluated in the gill and liver tissues of zebrafish (Danio rerio), exposed for 16days to a sublethal concentration (CL10) of two commercial detergents (laundry and dishwasher use) and an anionic surfactant: alkyl lauryl sulfonate (LAS). The results demonstrated high toxicity with dishwasher detergent, resulting in high lipid peroxidation levels (MDA malondialdehyde evaluation). No differences in MDA concentrations were found among fish exposed to laundry detergent and organisms exposed to LAS. In the genetic damage evaluation, significant differences in the number of cells with DNA strand breaks (comets) were observed: the fish exposed to dishwasher detergent presented the highest number of damaged cells (79%), in comparison with those exposed to other products (laundry and LAS) and the control group (8% damaged cells). The toxicity of dishwasher detergent (biological detergent containing enzymes and perfume) was higher than the value observed with LAS. Laundry detergent does not contain enzymes or perfume and its toxicity was similar to LAS. Since detergents are complex mixtures of different substances, in which additive and/or synergistic effects may occur, the deleterious effect caused by the dishwasher detergent was probably due to the combined effects of the ingredients of detergent.

Radioprobing the conformation of DNA in a p53-DNA complex

Purpose: The frequency of DNA strand breaks produced by the decay of Auger electron-emitting radionuclides is inversely proportional to the distance of DNA nucleotides from the decay site; and thus is very sensitive to changes in the local conformation of the DNA. Analysis of the frequency of DNA breaks, or radioprobing, gives valuable information about the local DNA structure. More than 10 years ago, we demonstrated the feasibility of radioprobing using a DNA-repressor complex with a known structure. Herein, we used radioprobing to study the conformation of DNA in complex with the tumor suppressor protein 53 (p53). Several structures of p53-DNA complexes have been solved by X-ray crystallography. These structures, obtained with the p53 DNA binding domain, a truncated form, laid the groundwork for understanding p53-DNA interactions and their relation to p53 functions. However, whether all observed stereochemical details are relevant to the native p53-DNA complex remains unclear. A common theme of the crystallographic structures is the lack of significant bending in the central part of the DNA response element. In contrast, gel electrophoresis and electron microscopy data showed strong DNA bending and overtwisting upon binding to the native p53 tetramer.Methods: To analyze DNA in complex with p53, we incorporated 125I-dCTP in two different positions of synthetic duplexes containing the consensus p53-binding site.Results: The most significant changes in the break frequency distributions were detected close to the center of the binding site, which is consistent with an increase in DNA twisting in this region and local DNA bending and sliding.Conclusions: Our data confirm the main results of the studies made in solution and lay a foundation for systematic examination of interactions between DNA and native p53 using 125I radioprobing.

Positioning Effects of KillerRed inside of Cells correlate with DNA Strand Breaks after Activation with Visible Light

Fluorescent proteins (FPs) are established tools for new applications, not-restricted to the cell biological research. They could also be ideal in surgery enhancing the precision to differentiate between the target tissue and the surrounding healthy tissue. FPs like the KillerRed (KRED), used here, can be activated by excitation with visible day-light for emitting active electrons which produce reactive oxygen species (ROS) resulting in photokilling processes. It is a given that the extent of the KRED's cell toxicity depends on its subcellular localization. Evidences are documented that the nuclear lamina as well as especially the chromatin are critical targets for KRED-mediated ROS-based DNA damaging. Here we investigated the damaging effects of the KRED protein fused to the nuclear lamina and to the histone H2A DNA-binding protein. We detected a frequency of DNA strand breaks, dependent first on the illumination time, and second on the spatial distance between the localization at the chromatin and the site of ROS production. As a consequence we could identify defined DNA bands with 200, 400 and (600) bps as most prominent degradation products, presumably representing an internucleosomal DNA cleavage induced by KRED. These findings are not restricted to the detection of programmed cell death processes in the therapeutic field like PDT, but they can also contribute to a better understanding of the structure-function relations in the epigenomic world.

Aristolochic acid suppresses DNA repair and triggers oxidative DNA damage in human kidney proximal tubular cells

Aristolochic acid (AA), derived from plants of the Aristolochia genus, has been proven to be associated with aristolochic acid nephropathy (AAN) and urothelial cancer in AAN patients. In this study, we used toxicogenomic analysis to clarify the molecular mechanism of AA-induced cytotoxicity in normal human kidney proximal tubular (HK-2) cells, the target cells of AA. AA induced cytotoxic effects in a dose-dependent (10, 30, 90 microM for 24 h) and time-dependent manner (30 microM for 1, 3, 6, 12 and 24 h). The cells from those experiments were then used for microarray experiments in triplicate. Among the differentially expressed genes analyzed by Limma and Ingenuity Pathway Analysis software, we found that genes in DNA repair processes were the most significantly regulated by all AA treatments. Furthermore, response to DNA damage stimulus, apoptosis, and regulation of cell cycle, were also significantly regulated by AA treatment. Among the differentially expressed genes found in the dose-response and time-course studies that were involved in these biological processes, two up-regulated (GADD45B, NAIP), and six down-regulated genes (TP53, PARP1, OGG1, ERCC1, ERCC2, and MGMT) were con-firmed by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). AA exposure also caused a down-regulation of the gene expression of anti-oxidant enzymes, such as superoxide dismutase, glutathione reductase, and glutathione peroxidase. Moreover, AA treatment led to increased frequency of DNA strand breaks, 8-hydroxydeoxyguanosine-positive nuclei, and micronuclei in a dose-dependent manner in HK-2 cells, possibly as a result of the inhibition of DNA repair. These data suggest that oxidative stress plays a role in the cytotoxicity of AA. In addition, our results provide insight into the involvement of down-regulation of DNA repair gene expression as a possible mechanism for AA-induced genotoxicity.

Imidazopurinones are markers of physiological genomic damage linked to DNA instability and glyoxalase 1-associated tumour multidrug resistance

Glyoxal and methylglyoxal are reactive dicarbonyl metabolites formed and metabolized in physiological systems. Increased exposure to these dicarbonyls is linked to mutagenesis and cytotoxicity and enhanced dicarbonyl metabolism by overexpression of glyoxalase 1 is linked to tumour multidrug resistance in cancer chemotherapy. We report herein that glycation of DNA by glyoxal and methylglyoxal produces a quantitatively important class of nucleotide adduct in physiological systems—imidazopurinones. The adduct derived from methylglyoxal-3-(2′-deoxyribosyl)-6,7-dihydro-6,7-dihydroxy-6/7-methylimidazo-[2,3-b]purine-9(8)one isomers—was the major quantitative adduct detected in mononuclear leukocytes in vivo and tumour cell lines in vitro. It was linked to frequency of DNA strand breaks and increased markedly during apoptosis induced by a cell permeable glyoxalase 1 inhibitor. Unexpectedly, the DNA content of methylglyoxal-derived imidazopurinone and oxidative marker 7,8-dihydro-8-oxo-2′-deoxyguanosine were increased moderately in glyoxalase 1-linked multidrug resistant tumour cell lines. Together these findings suggest that imidazopurinones are a major type of endogenous DNA damage and glyoxalase 1 overexpression in tumour cells strives to counter increased imidazopurinone formation in tumour cells likely linked to their high glycolytic activity.

Leukocyte DNA damage in children with iron deficiency anemia: effect of iron supplementation

Iron deficiency is frequently associated with anemia. Iron is a transition-metal ion, and it can induce free radical formation, which leads to formation of various lesions in DNA, proteins, and lipids. The aim of this study was to investigate baseline oxidative DNA damage and to clarify the role of the administration of a therapeutic dose of iron on DNA oxidation in children with iron deficiency anemia (IDA). Twenty-seven children with IDA and 20 healthy children were enrolled in the study. Leukocyte DNA damage (strand breaks and Fpg-sensitive sites) was assessed using comet assay before and after 12 weeks of daily iron administration. Before the iron administration, the frequency of DNA strand breaks in the children with IDA was found to be lower than those in the control group (P < 0.05), but there was not a significant difference for frequency of Fpg-sensitive sites. After 12 weeks of iron administration, the frequency of both DNA strand breaks and Fpg-sensitive sites were found to be increased (P < 0.01). No significant association was determined between DNA damage parameters and hemoglobin, hematocrit, serum iron, total iron binding capacity, and ferritin. In conclusion, basal level of DNA strand breaks is at a low level in children with IDA. After iron administration, DNA strand breaks and Fpg-sensitive sites, which represent oxidatively damaged DNA, increased. However, this increase was unrelated to serum level of iron and ferritin.

Assessment of DNA damage and plasma catalase activity in healthy term hyperbilirubinemic infants receiving phototherapy

Phototherapy (PT) is the most widely used form of treatment for unconjugated hyperbilirubinemia. One possible harmful consequence of PT is of a genetic nature. High levels of bilirubin may lead to oxidative damage in newborns: photochemical reactions may produce toxic photoproducts, probably peroxides. In order to investigate this hypothesis further under in vivo conditions, DNA strand-break frequency was examined by means of the comet assay in peripheral lymphocytes of icteric newborns undergoing PT treatment, and the levels of catalase, an antioxidant enzyme, were determined. We analyzed 20 term non-hemolitic hyperbilirubinemic jaundiced neonates before PT (‘before PT’ group) and just prior to ending PT (‘after PT’ group) and compared comet scores of these patients with those of 20 healthy term neonates who all had bilirubin levels in the physiological range. Comet scores (tail length, tail moment and %DNA in tail) of the group ‘before phototherapy’ were 23.5 ± 16.3, 7.41 (0.97–40.7), 33.0 ± 12.1, respectively and scores of after phototherapy group were 3.2 ± 1.8, 0.29 (0.3–3.2), 10.7 ± 3.7, respectively. Comet scores of the control group were 3.0 ± 2.9, 0.25 (0.03–3.22), 10.9 ± 4.5, respectively. Comet scores and plasma catalase activities in hyperbilirubinemic newborns were significantly higher before phototherapy, compared with the values after phototherapy and in the control groups ( p < 0.001). There was no statistical difference between the ‘after phototherapy’ group and the controls ( p > 0.05). These results indicate that high serum bilirubin level has genotoxic effects as is evident from the high rate of DNA strand-breaks in jaundiced newborns. Also PT does not cause an increase in DNA oxidation or induce the genotoxic effects of bilirubin. The counteracting effect of higher catalase activities in hyperbilirubinemic newborns may be responsible for the inactivating toxic and DNA-damaging effects of PT.

Disruption of the Plasmodium berghei 2-Cys peroxiredoxin TPx-1 gene hinders the sporozoite development in the vector mosquito

To investigate the physiologic role of cytosolic 2-Cys peroxiredoxin of Plasmodium berghei (PbTPx-1), we infected the vector mosquito Anopheles stephensi with a parasite carrying a targeted knockout of pbtpx-1 (Prx-KO). The number of Prx-KO midgut oocysts at 14–15 days post-feeding (pf) was comparable to that of the parent strain (WT); however, the numbers of sporozoites that formed in midgut oocysts and accumulated in the salivary gland of Prx-KO-infected mosquitoes by 21 days pf were decreased to 10–20% and 3–10%, respectively, of those values in WT-infected mosquitoes. A higher frequency of DNA strand breaks was detected in Prx-KO oocysts than in WT oocysts. Sporozoites carrying the targeted disruption had reduced infectivity in mice; however, the knockout did not affect the ability of the sporozoite to reach the liver parenchyma and initiate exo-erythrocytic form (EEF) development. TPx-1 may be involved in development during exponentially multiplying stages, such as sporozoites and EEF.

Ambient and Biological Monitoring of Exposure and Genotoxic Effects in Mastic Asphalt Workers Exposed to Fumes of Bitumen

Mastic asphalt workers may be exposed to polycyclic aromatic hydrocarbons (PAH) present in bitumen. We conducted a cross-shift study to determine genotoxic effects after exposure to bitumen. For this purpose, external and internal exposure of 202 mastic asphalt workers exposed to bitumen and 55 construction workers without exposure to bitumen was assessed. Exposure by inhalation to fumes of bitumen during the shift was measured by personal ambient monitoring. To assess overall exposure to bitumen (by inhalation and dermal absorption), 1-hydroxypyrene (1-OHP) and the sum of 1-, 2 + 9-,3-,4-hydroxyphenanthrene (OHPhe) were determined in pre- and postshift urine. 8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) adducts, anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (anti-BPDE) DNA adducts, DNA strand breaks and alkali-labile sites, and micronucleus frequencies were determined as biomarkers of genotoxic effects in white blood cells (WBC). Concentrations of fumes of bitumen were correlated with a moderate association with 1-OHP and OHPhe after work shift (r s = 0.25, P < 0.001 and r s = 0.36, P < 0.001). Significantly increased 8-oxodGuo adduct levels were observed after shift in both exposure groups (P < 0.0001). Paradoxically, decreased DNA strand break frequencies were observed after shift in both groups (P < 0.05). Postshift values in DNA strand break frequency were associated with 1-OHP (Spearman rank correlation coefficient 0.19, P = 0.01). Significantly more 8-oxodGuo adducts and DNA strand breaks were found in workers exposed to bitumen before and after shift compared with the reference group. However, no dose-dependent association was observed between exposure and genotoxic effects. Nevertheless, the findings indicate that workers exposed to bitumen exerted a higher level of DNA damage (8-oxodGuo and DNA strand breaks) in WBC compared with reference subjects.

Induction of DNA strand breaks and oxidative stress in HeLa cells by ethanol is dependent on CYP2E1 expression

Induction of cytochrome P4502E1 (CYP2E1) is considered to be an important mechanism by which ethanol can cause toxicity related to oxidative stress both in vivo and in vitro. In the current study, we used HeLa cells with doxycycline-regulated CYP2E1 expression to test the hypothesis that induction of CYP2E1 could lead to secondary DNA oxidation that could potentially contribute to the carcinogenicity of ethanol in vivo. Overexpression of CYP2E1 protein was not associated with oxidative stress per se as assessed by markers of lipid peroxidation (cis-parinaric acid oxidation), glutathione depletion and elevation of intracellular reactive oxygen species (dichlorofluoroscin oxidation) in the presence or absence of ethanol substrate (10 mM, 24 h). Furthermore, there was no evidence of elevation of frequency of DNA strand breaks as assessed by the comet assay. In contrast, however, after pre-incubation of cells with L-buthionine-(S,R)-sulphoximine (BSO, 10 microM) which caused a 75% reduction in intracellular reduced glutathione (GSH) levels, CYP2E1 expression resulted in oxidative stress as assessed by all of these markers and DNA strand breaks but only in the presence of ethanol (10 mM). No effect was observed under these conditions in control cells not expressing CYP2E1. Furthermore, these effects could be attenuated by co-incubation with 1-aminobenzotriazole (0.5 mM), a suicide inhibitor of P450 activity. In conclusion, in this in vitro model CYP2E1-mediated interaction with ethanol results in the intracellular oxidative stress and the formation of DNA strand breaks which are detectable in cells pre-sensitized by depletion of intracellular levels of GSH.

14.6 mT ELF magnetic field exposure yields no DNA breaks in model systemSalmonella, but provides evidence of heat stress protection

In this study, we demonstrate that common extremely low frequency magnetic field (MF) exposure does not cause DNA breaks in this Salmonella test system. The data does, however, provide evidence that MF exposure induces protection from heat stress. Bacterial cultures were exposed to MF (14.6 mT 60 Hz field, cycled 5 min on, 10 min off for 4 h) and a temperature-matched control. Double- and single-stranded DNA breaks were assayed using a recombination event counter. After MF or control exposure they were grown on indicator plates from which recombination events can be quantified and the frequency of DNA strand breaks deduced. The effect of MF was also monitored using a recombination-deficient mutant (recA). The results showed no significant increase in recombination events and strand breaks due to MF. Evidence of heat stress protection was determined using a cell viability assay that compared the survival rates of MF exposed and control cells after the administration of a 10 min 53 degrees C heat stress. The control cells exhibited nine times more cell mortality than the MF exposed cells. This Salmonella system provides many mutants and genetic tools for further investigation of this phenomenon.

DNA strand breaks in the lymphocytes of workers exposed to diisocyanates: indications of individual differences in susceptibility after low-dose and short-term exposure

Diisocyanates are chemically reactive and induce asthma, but data on genotoxic effects of diisocyanates in humans are limited. The investigation presented here used short term diisocyanate chamber exposure to study DNA strand breaks in lymphocytes of 10 healthy individuals and of 42 workers, with airway symptoms, who had previously been exposed to diisocyanates. The alkaline version of the Comet assay was used to analyse DNA strand breaks in lymphocytes. In addition, blood samples of 10 further control individuals without any exposure to diisocyanates were studied. Substances studied were 4,4'-methylenediphenyldiisocyanate (MDI, n=25), 2,4-toluenediisocynate and 2,6-toluenediisocyanate (TDI, n=5), and 1,6-hexamethylenediisocyanate (HDI, n=12), at concentrations between 5 and 30 ppb for 2 h. Lymphocytes isolated from the subjects before exposure and 30 min and 19 h after were used to evaluate DNA damage. No significant changes in DNA strand-break frequencies were measured, as Olive tail moment (OTM), either between groups or before and after diisocyanate exposure. OTM was similar in subjects with an asthmatic reaction (MDI, n=5; TDI, n=1; HDI, n=1) and in subjects without such a reaction. However, a small and susceptible group (about 10% of the individuals studied) could be identified with higher frequencies of DNA strand breaks in lymphocytes after chamber exposure. The occurrence of DNA damage in this group may be based on indirect mechanisms such as oxidative stress or apoptosis.

Genotoxicity of the herbicide 2,4-dichlorophenoxyacetic and a commercial formulation, 2,4-dichlorophenoxyacetic acid dimethylamine salt. I. Evaluation of DNA damage and cytogenetic endpoints in Chinese Hamster ovary (CHO) cells

Genotoxicity of the 2,4-dichlorophenoxyacetic acid (2,4-D) and a commercially-used derivative, 2,4-D dimethylamine salt (2,4-D DMA), was evaluated in CHO cells using SCE and single cell gel electrophoresis (SCGE) assays. Log-phase cells were treated with 2.0–10.0 μg/ml of herbicides and harvested 24 and 36 h later for SCE analysis. Both agents induced significant dose-dependent increases in SCE, regardless of the harvesting time (2,4-D: r = 0.98 and r = 0.88, P < 0.01, for 24 and 36 h harvesting times; 2,4-D DMA: r = 0.97 and r = 0.88, P < 0.01, for 24 and 36 h harvesting times). Neither test compound altered cell-cycle progression or proliferative replication index ( P > 0.05), but the higher doses of both compounds reduced the mitotic index of cultures harvested at 24 and 36 h ( P < 0.05). A 90-min treatment with 2.0–10.0 μg/ml 2,4-D and 2,4-D DMA produced dose-dependent increases in the frequency of DNA-strand breaks detected in the SCGE assay, both in cultures harvested immediately after treatment and in cultures harvested 36 h later. The doses of 2,4-D and 2,4-D DMA were equally genotoxic in all of the assays. The results indicate that 2,4-D induces SCE and DNA damage in mammalian cells, and should be considered as potentially hazardous to humans.